The generation of protective immunity that reduces or prevents re-infection with the same pathogen is a hallmark of the adaptive immune system. One key component of this process is the formation of memory T cells. Several factors are known to impact the numbers, as well as the effectiveness of the memory T cells generated in response to virus infection. These include antigen abundance and duration, cytokine signals, and costimulatory molecules. However, the precise molecular mechanisms by which the integration of these signals regulate memory cell formation and function are not well understood. In particular, there is remarkably little known about how differences in TCR signaling, due to variations in TCR affinity/avidity for antigen-MHC complexes, impact the differentiation of effector versus memory CD8+ T cells at the molecular level. Our work has focused on the signaling pathways downstream of the TCR. We have found that the transcription factor, IRF4, is upregulated upon TCR stimulation of nave T cells, and that the amount of IRF4 produced in each T cell is dependent on the strength of the TCR signal. We also found that IRF4 upregulation was markedly impaired in T cells lacking the Tec kinase, ITK, a known modulator of TCR signal strength. Stimulation of IRF4- deficient CD8+ T cells induced high levels of Eomesodermin, a transcription factor associated with memory CD8+ T cells. Together, these data lead us to hypothesize that strong TCR signaling in CD8+ T cells responding to a virus infection induces robust ITK activation and high expression of IRF4. In turn, these factors induce high expression of T-bet and Blimp-1, suppress TCF1 and Eomesodermin expression, and thereby promote a vigorous expansion of short-lived effector cells. In contrast, cells receiving weak TCR stimulation would activate little ITK, upregulate low levels of IRF4, and differentiate rapidly into memory precursor cells. To test this hypothesis, we will examine the CD8+ T cell response to LCMV-Armstrong, as well as Influenza A virus, by cells carrying two, one, or zero copies of a functional IRF4 gene (IRF4+/+, IRF4+/-, IRF4-/-). We will also modulate the strength of TCR signaling by infecting wild type mice with LCMV- Armstrong, followed by varying doses of a small molecule inhibitor of ITK. As a third approach, we will use an LCMV-Armstrong variant carrying a point mutation in the GP33 peptide epitope recognized by the P14 TCR, leading to low affinity stimulation of P14 transgenic CD8+ T cells. In the second aim, we will address a set of putative downstream targets of IRF4, and will assess whether TCF1 is involved in the mechanism by which IRF4 represses Eomesodermin expression. In the third aim, we will examine the CD8+ T cell responses of mice carrying heterozygous mutations in ITK, IRF4, or both, to infections of the clone 13 strain of LCMV, a virus that establishes chronic infections in wild type mice. In the fourth aim, we will determine whether IL-4 synergizes with low TCR signal strength to promote memory T cell differentiation in vivo. Together, these studies will provide important insights into the signaling pathways that regulate short-lived effector versus memory precursor T cell formation, and in particular, the role of TCR signaling in this process.